Chromatin contains information beyond the DNA sequence that is needed to regulate the genome and allow the proper development of multi-cellular organisms. Posttranslational modifications (PTMs) of histones modulate the organization of chromatin for replication, repair and transcription of genetic information, and are hypothesized to be the carriers of information that is inherited 'epigenetically' over multiple cel generations. Importantly, investigations into histone PTM function in multicellular eukaryotes have been limited by pleiotropic effects resulting from pharmacological and genetic manipulation of epigenetic regulatory proteins that create, remove, or respond to histone PTMs. Because of this limitation, there is a gap in knowledge regarding the role of individual histone residues as carriers of information. The objective of this proposal is to generate a comprehensive experimental platform in Drosophila melanogaster for spatial and temporal manipulation of post-translationally modified histone residues during animal development. To study the biological function of a specific histone PTM, the acceptor residue must be changed to an amino acid that cannot be appropriately modified. Then, all wild-type copies of that histone gene must be replaced with the mutant copy. We will use a transgenic strategy for replacement of the entire Drosophila histone multigene family with engineered gene clusters that express specific histone mutants, and create experimental tools for the temporal and spatial restriction of these mutations. These studies will directly test the roles of post- translationally modified histone residues during animal development, and will create a sustainable and expandable resource for the study of epigenetic phenomena in metazoans. Because disruption of evolutionarily conserved histone PTMs is thought to underlie many human diseases, including cancer, this research will directly impact human health.